Tetanus toxin is internalized by way of acidic endosomes. Monensin and bafilomycin, agents which disrupt the endosomal pH gradient, protect neurons against the effects of the toxin. In synaptic terminals poisoned with tetanus toxin, there are twice as many synaptic vesicles aligned at the active zone as there are in control preparations. This finding strengthens the notion that the toxin acts by preventing vesicle fusion with the presynaptic membrane. Tetanus toxin acts with a different time course on inhibitory and excitatory synapses. Following exposure to tetanus toxin, inhibitory synapses are blocked more quickly and recover more slowly than excitatory synapses. Neurotransmitter release from neuronal cell cultures has proven to be a reliable assay for the efficacy of anthrax toxin-tetanus toxin fusion proteins.